1. Field of the Invention
The present invention relates to a novel DNA, more particularly, to a novel DNA which encodes trehalase, and uses thereof.
2. Description of the Prior Art
Trehalose, a non-reducing disaccharide which consists of glucose units as constituent saccharide, is widely distributed in the natural world, for example, in bacteria, fungi, algae, insects, Crustacean, etc. In organisms such as insects which have a relatively-large amount of trehalose in their bodies, trehalose would play an important role as energy source and relate to the maintenance of physiological function such as cold resistance. Mammals including humans have long been utilizing trehalose widely from mushrooms, seaweeds, fermented foods, etc, as reported by Oku et al., in xe2x80x9cJournal of The Japanese Society For Food Science And Technologyxe2x80x9d, Vol. 45, No. 6, pp. 381-384 (1998). As disclosed in Japanese Patent Kokai Nos. 143,876/95 and 213,283/95 applied for by the present applicant, the establishment of technologies for industrial-scale production of trehalose has more increased the interest of trehalose in the maintenance and regulation of biological functions in mammals, and this results in energetic and continuous researches in various fields.
Trehalase is an enzyme which specifically hydrolyzes the glucosidic bond in trehalose. Because of this substrate specificity, the enzyme may deeply correlate to the trehalose level in vivo in organisms such as insects and relate to the regulation of their biological functions. Even in mammals with no significant amount of trehalose, trehalase is found in animals such as humans, mice and rats. Major physiological role of trehalase in mammals would be the hydrolysis of externally-ingested trehalose when the saccharide is digested and absorbed by the mammals. It was reported that trehalase is commonly found in specific mammalian organs independently of the intake of trehalose, and hence there still remains many unknown biological roles of trehalase per se. As described above, the roles of mammalian trehalase in the maintenance and regulation of their physiological functions have also been focused recently, along with the increasing interest in trehalose.
Methods in a molecular biological manner are very useful for elucidating the physiological roles of specific enzymes or polypeptides in living bodies. Mice are the animals commonly used widely as models for elucidating the biological functions of mammals including humans. Thus, the techniques and analyses for murine trehalase in a molecular biological manner would be particularly useful for elucidating the physiological roles of mammalian trehalase. Any nucleotide sequence of murine trehalase, which is requisite for its molecular biological engineering, has not been elucidated, and any structure of the enzyme per se has not been disclosed. Urgently expected are as follows: The elucidation of a nucleotide sequence of a DNA for murine trehalase, the establishment of a DNA useful for engineering the enzyme in a molecular biological manner, and uses thereof.
In view of the foregoing, the object of the present invention is to provide a DNA useful for engineering murine trehalase in a molecular biological manner, and uses thereof.
To attain the above object, the present inventors widely screened cDNAs for RNAs, collected from mice, to isolate a cDNA for murine trehalase. As a result, the present inventors obtained from a murine intestine a cDNA which consists of about 2,000 base pairs (hereinafter abbreviated as xe2x80x9cbpxe2x80x9d) and expresses a polypeptide with trehalase activity. When compared with nucleotide sequences of conventionally known DNAs, the cDNA contained, as a coding sequence of the polypeptide, a nucleotide sequence of SEQ ID NO: 1 which was clearly different from conventionally known DNAs. The present inventors also found the fact that a gene for the cDNA exists on a murine chromosome. Based on these results, they confirmed that the cDNA was for murine trehalase, and then analyzed murine genomic DNAs with reference to the nucleotide sequence of the cDNA, elucidated the structure of a gene for murine trehalase which consists of a total length of about 20,000 bp and comprises the nucleotide sequence of SEQ ID NO: 1 and introns for splitting the nucleotide sequence, and isolated the gene as a genomic DNA. The present inventors also confirmed that a part or the whole of the isolated DNA can be arbitrarily used to engineer and analyze murine trehalase in a molecular biological manner. For example, such a DNA can be advantageously used to prepare transformants suitable for producing polypeptides used as murine trehalase standard specimens and as antigens for preparing anti-murine trehalase antibodies, and to prepare transgenic animals and trehalase gene knockout animals. The present invention is based on these findings.
The present invention solves the above object by providing a DNA which comprises a part or the whole of the nucleotide sequence of SEQ ID NO: 1 that encodes trehalase, a polypeptide obtainable by the expression of the DNA, a process for producing the polypeptide using the DNA, and a transgenic- and knockout-animals obtainable therewith.
The present invention relates to a novel DNA which encodes trehalase and uses thereof. The term xe2x80x9ctrehalasexe2x80x9d as referred to in the present invention means an enzyme, i.e., a protein or polypeptide which specially hydrolyzes the glucosidic bond in xcex1,xcex1-trehalose, and the hydrolysis activity is called xe2x80x9ctrehalase activityxe2x80x9d in the present invention. The DNA of the present invention comprises a part or the whole of the nucleotide sequence of SEQ ID NO: 1 which encodes trehalase, and usually it is mouse origin.
In the present invention, the term xe2x80x9ca part or the whole of the nucleotide sequence of SEQ ID NO: 1xe2x80x9d generally means xe2x80x9ca nucleotide sequence which contains at least ten and several contiguous bases in the nucleotide sequence of SEQ ID NO: 1.xe2x80x9d
Preferred examples of the DNA of the present invention include DNAs as cDNAs which comprise a part or the whole of the nucleotide sequence of SEQ ID NO: 1, DNAs as genomic DNAs obtainable by fragmenting chromosomes, and other DNAs obtainable by applying replacement, deletion, and/or addition of bases to the above DNAs. These DNAs do not contain telomeres as a characteristic structure in the terminal region of chromosomes of eukaryotic cells such as mammalian cells, and such DNAs are usually provided in the from of isolated liner DNAs which are composed of not more than about 20,000 bp and which are distinguishable from naturally-occurring chromosomal DNAs and RNAs present in mammalian cells. All of these DNAs according to the present invention may be homologous to the nucleotide sequences of cDNAs for human and rat trehalases, registered in xe2x80x9cGenBankxe2x80x9d, a database of nucleic acids provided by the National Institute of Health of USA, under the accession Nos. AB000824 and AF038043, respectively, but are not completely coincided with the above registered nucleotide sequences.
A cDNA, as an example of the DNA of the present invention, usually comprises a part or the whole of the nucleotide sequence of SEQ ID NO: 1 as a coding sequence or nucleotide sequence which encodes the polypeptide of the present invention, and may contain a nucleotide sequence as a non-coding region at the 5xe2x80x2 and/or 3xe2x80x2 end regions. Usually, such a cDNA can be obtained by preparing in the usual manner a cDNA using a RNA as a template obtainable from organs such as intestines, kidneys, livers, and lungs from mice or their relative rodents, and cell lines established from these organs; and screening the cDNA with an index of the existence of annealing with at least a part of the nucleotide sequence of SEQ ID NO: 1. Any method such as PCR, colony hybridization, or plaque hybridization method commonly used in this art can be used as the screening method. Although the DNAs thus obtained are varied depending on the nature of species, strains, individuals, and organs as RNA sources, such DNAs are clearly homologous to the nucleotide sequence of SEQ ID NO: 1, usually they have a homology of over 94%, more desirably, 97% or higher. For example, as described in the later described examples, a DNA, which has the nucleotide sequence of SEQ ID NO: 3 and includes the nucleotide sequence of SEQ ID NO: 1, is generally obtained by using RNAs from ddY murine intestines as materials.
A genomic DNA, as another form of the DNA of the present invention, generally contains a part or the whole of the nucleotide sequence of SEQ ID NO: 1 as a coding region, and another nucleotide sequence as an intron and non-translated region. For example, the genomic DNA can be obtained by applying PCR using, as templates, chromosomes obtainable from appropriate organs and cells of mice and their relative rodents; and oligonucleotides as primers prepared based on the nucleotide sequence of SEQ ID NO: 1; or by engineering genomic libraries from the chromosomes in the usual manner, and screening the desired DNAs using a probe prepared based on the nucleotide sequence of SEQ ID NO: 1. Depending on the nature of species, strains, individuals, and organs of the sources of chromosomes, the DNAs thus obtained may be varied in some degrees with respect to the nucleotide sequence of a coding sequence, non-translated region, and/or intron. The DNAs, however, have a clear homology to the nucleotide sequence of SEQ ID NO: 1, usually, a homology of over 94%, more preferably, 97% or higher. For example, a chromosome from ICR Swiss mouse provides a DNA which comprises both the nucleotide sequence of SEQ ID NO: 1 and at least an intron that exists between the bases 80 and 81 in the nucleotide sequence. From a chromosome of C57BL/6 mouse, the following DNA can be obtained: A DNA which comprises the nucleotide sequence of SEQ ID NO: 1 and at least the introns which exist respectively between the bases 181 and 182, 326 and 327, 414 and 415, 515 and 516, 608 and 609, 725 and 726, 848 and 849, 898 and 899, 1093 and 1094, 1311 and 1312, 1423 and 1424, 1536 and 1537, and 1590 and 1591 in the nucleotide sequence of SEQ ID NO: 1. The above genomic DNA according to the present invention can be usually obtained as a liner DNA which consists of about 20,000 bases or fewer.
As described above, the isolated DNA of the present invention can be made modifications such as fragmentation, replacement, deletion, and/or addition of bases by conventional methods used in this art. The DNA of the present invention includes the aforesaid modified DNAs as long as they contain at least a part of the nucleotide sequence of SEQ ID NO: 1. For example, single-stranded DNAs or oligonucleotides, which consist of at least ten and several contiguous bases in the sense or anti-sense strand of the above DNAs, are useful as PCR primers and hybridization probes to detect or amplify the DNA of the present invention. The oligonucleotides can also be used to detect and amplify trehalase-related DNAs, which comprise a part of the nucleotide sequence of SEQ ID NO: 1, other than those illustrated in the present specification. The oligonucleotides as PCR primers can be incorporated with other nucleotide sequences such as restriction-enzyme-recognizing sites, and modified by replacement, deletion, and/or addition of bases with or without altering the amino acid sequence encoded by the oligonucleotides. The DNAs as PCR primers, which have been received with any of the above modifications, can be arbitrarily used to prepare recombinant DNAs as expression vectors for expressing trehalase and as targeting vectors for preparing knockout mice. These DNAs in the form of oligonucleotides can be obtained by chemical-synthetic-methods which are commonly used in this art.
By applying conventional PCR reaction to the above DNAs, the following DNAs can be obtained: DNAs which consist essentially of at least a part of the coding sequences for the above exemplified DNAs, for example, those which consist of either the nucleotide sequence of SEQ ID NO: 1 or bases 58 to 1728 in the nucleotide sequence of SEQ ID NO: 1, and nucleotide sequences which are partial nucleotide sequences of SEQ ID NO: 1 and which correspond to amino acid sequences that consist of at least ten contiguous amino acids. For example, by using the recombinant DNA technology, these DNAs can be arbitrarily used to produce murine trehalase-related polypeptides used as standard specimens for qualitative- and quantitative-analyses of murine trehalase, and as antigens for preparing anti-murine trehalase antibodies. In the case of expressing the aforesaid DNAs, replacement, addition, and/or deletion of bases can be further introduced into a part of the DNAs, depending on the hosts used. Examples of such modifications are as follows:
(i) Replacement of a part of the bases of the DNAs with reference to the frequency of codons used in host cells without altering the inherent amino acid sequences encoded by the DNAs;
(ii) Addition of initiation and termination condons to the DNAs;
(iii) Addition of nucleotide sequences recognizable by specific substances such as histidine tag to the N- or C-terminus of polypeptides to be expressed;
(iv) Deletion of a part of the bases of the DNAs in the 5xe2x80x2 or 3xe2x80x2 end region to increase the efficiency of DNA expression; and
(v) Insertion of one or more of the above introns into suitable sites in the DNAs.
These DNAs of the present invention comprise the nucleotide sequences which usually have a homology of over 94%, more preferably, 97% or higher to the sequence which consists of at least about 30 contiguous bases in the nucleotide sequence of SEQ ID NO: 1. The DNA of the present invention includes those in the form of a recombinant DNA and those introduced into appropriate host cells.
The present invention provides a polypeptide obtainable by the expression of any of the above DNAs. The polypeptide of the present invention comprises an amino acid sequence which has a homology of 94% or higher, more preferably, 97% or higher to an amino acid sequence which consists of ten and several contiguous amino acids in the amino acid sequence of SEQ ID NO: 2: and may have trehalase activity. For example, an amino acid sequence, which consists of about 20 amino acids in the N-terminal region of the amino acid sequence of SEQ ID NO: 2, is capable of functioning as signal peptide; and another animo acid sequence, which consists of amino acids 20 to 576 in the amino acid sequence where the signal peptide has been eliminated, can participate in the expression of trehalase activity. The polypeptide of the present invention can be obtained in a desired amount by the following process of the present invention, which comprises the steps of producing the polypeptide from cells capable of producing the polypeptide of the present invention, i.e., a polypeptide which comprises at least a part of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1; and collecting the produced polypeptide. Transformants, which have been introduced with any of the above DNAs that consist essentially of at least a part of the coding sequence, are particularly useful as the cells used in the process of the present invention. These transformants can be usually obtained by annealing any of the above DNAs with autonomously-replicable vectors into recombinant DNAs, and introducing the DNAs into appropriate hosts. The autonomously-replicable vectors can be selected from conventional plasmid vectors such as pCDM8, pcDNAI/Amp, pcDL-SR xcex1, BCMGSNeo, pSV2-neo, pSV-2gpt, pEF-BOS, pCEV4, pME18S, pKY4, pKK223-3, pVL1392, and pVL1393. In general, the replicable vectors contain appropriate nucleotide sequences, which allow the DNA of the present invention to express in each hosts, such as promoters, enhancers, replication origins, termination sites, and splicing- and/or selective-sequences. Any method conventionally used in this art can be selectively used to ligate the DNA of the present invention to the above vectors. For example, addition of linkers or sequences for recognizing restriction enzymes by PCR, and treatment of restriction enzymes or ligases are all useful.
Any cells derived from microorganisms, plants, and animals including vertebrates such as mammals and invertebrates such as insects, which are all commonly used in this art to prepare transformant cells, can be used as host cells to be introduced with the DNA of the present invention. To prepare the polypeptide of the present invention at a lesser cost and in a relatively-high yield, microorganisms such as Escherichia coli and Bacillus subtilis, and cells from insects can be preferably used as hosts. Eukaryotic cells from yeasts and animals can be preferably used when the polypeptide is directed to use in the field of reagents for researches or pharmaceuticals for mammals which require a polypeptide equivalent to the one present naturally in the murine body with respect to saccharide chains to be added to and intra- and extra-cellar locations of the polypeptide. Examples of such host cells from animals include COS-1 cells (ATCC CRL-1650), CHO-K1 cells (ATCC CCL-61), 3T3-Swiss albino cells (ATCC CCL-92), C127I cells (ATCC CRL-1616), CV-1 cells (ATCC CCL-70), HeLa cells (ATCC CCL-2), MOP-8 cells (ATCC CRL-1709), and variants thereof; epithelial cells from humans, monkeys, mice, and hamsters; stromal cells; hematopoietic cells; and cells from insects such as Sf9 cells, commercialized by BD PharMingen, 10975 Torreyana Road, San Diego, Calif. 92121, USA, and High Five cells, commercialized by Invitrogen BV, NV Leek, Netherlands. To introduce the DNA of the present invention into the above host cells, any of the following conventional methods can be used; DEAE-dextran method, calcium phosphate method, electroporation, lipofection method, microinjection method, and virus infection method using retrovirus, adenovirus, herpes virus, or vaccinia virus. Desired clones can be selected from the resulting transformants with an index of the existence of an introduced DNA or the productivity of the polypeptide. With regard to the aforesaid recombinant DNAs and transformants, materials and methods commonly used in this art are described in detail in xe2x80x9cCurrent Immuno-protocol in Molecular Biologyxe2x80x9d, chapters 1-9 and 15-16 (1996), edited by Frederick M. Ausubel et al., published by John Wiley and Sons Inc., New York, U.S.A.
The transformants thus obtained produce the polypeptide of the present invention intra- or extra-cellularly by culturing or proliferating in media under the conditions selected depending on the type of host cells or the structure of vectors used to introduce the DNA of the present invention. Although, the produced polypeptide can be used intact to suit to final use, it is usually purified before use. For the purification, any conventional methods commonly used in this art can be used, for example, salting out, dialysis, filtration, concentration, fractional precipitation, ion-exchange chromatography, gel filtration chromatography, absorption chromatography, isoelectric chromatography, hydrophobic chromatography, reverse phase chromatography, affinity chromatography, gel electrophoresis, and isoelectric electrophoresis. The polypeptide of the present invention, purified to a desired level, can be obtained by allowing the resulting fraction, which has been purified by the above purification methods, to examine and analyze properties such as amino acid sequence, molecular weight, and trehalase activity of the polypeptide; and collecting a fraction which exhibits the desired properties.
In this art, once a desired DNA is obtained, it can be commonly introduced into appropriate animals to obtain so called transgenic animals. The present invention also provides transgenic animals by applying conventional methods to the DNA of the present invention. The transgenic animals can be generally prepared by introducing the DNA, which comprises at least a part of the aforesaid coding sequence, into appropriate vectors which are selected depending on the species of the hosts used, in combination with another desired nucleotide sequences for promotors or enhancers, etc., if necessary; and introducing the resulting recombinant DNA into fertilized eggs or embryonic stem cells of host animals by either conventional methods such as microinjection or infection with viruses. The following animals can be advantageously used as host animals in the present invention because they can be bred easily: Rodents such as mice, rats, and hamsters which are used frequently as experimental animals; and other mammals such as goats, sheep, pigs, and cows which are commonly bred as domestic animals. The obtained cells introduced with the DNAs are transplanted to uterines or uterus of female animals in pseudopregnancy, which are the same species as the cells. Transgenic animals introduced with the DNA of the present invention can be obtained by applying hybridization or PCR method to newborns born by natural delivery or Cesarean section, and selecting animals introduced with the DNA. With these methods, the productivity of the polypeptide with trehalase activity of the present invention can be imparted to a desired animal. The transgenic animals thus obtained can be arbitrarily used in the production of the polypeptide of the present invention and also used as animal models for examining the in vivo influence of the polypeptide in living bodies, and used as animals for screening therapeutic, prophylactic, and diagnostic agents for mammalian diseases related to the excessive production of the polypeptide. The techniques for preparing transgenic animals as mentioned above are described in detail in xe2x80x9cNew Genetic Engineering Handbookxe2x80x9d, pp. 269-276 (1996), edited by Masami MURAMATSU, Hiroto OKAYAMA, and Masashi YAMAMOTO, published by Yodo Co., Ltd., Tokyo, Japan.
In this field, once the gene structure of a desired gene is revealed and a DNA which contains at least a part of the gene is isolated, animals with artificially destroyed genes, i.e., knockout animals can be generally obtained. A general preparation method of a knockout animal is described in the below with reference to a knockout mouse:
(i) Preparing a vector (targeting vector) to destroy a desired gene;
(ii) Introducing the targeting vector into murine embryonic stem cells (ES cells) with totipotency;
(iii) Selecting ES cells where the desired gene has been destroyed by the introduced targeting vector;
(iv) Infecting the selected ES cells into a murine blastula, transplanting the murine blastula to an expedient mouse, and selecting a chimaera mouse from newborns delivered from the expedient mouse;
(v) Inbreeding a male chimera mouse with a female wild-type mouse, and selecting a male and female heterozygotes from F1 mice delivered from the female mouse; and
(vi) Inbreeding the male and female heterozygotes, and selecting a homozygote (knockout mouse) from F2 mice delivered from the female mouse.
As mentioned above, since the present invention discloses a DNA as a genomic DNA which corresponds to a trehalase gene that comprises a part or the whole of the nucleotide sequence of SEQ ID NO: 1, knockout animals with a destroyed trehalase gene can be prepared by using the DNA in such a form. The present invention also provides such knockout animals. In the preparation of knockout animals of the present invention, targeting vectors used in the above step (i) are prepared. The targeting vector used in the present invention is generally prepared by applying replacement, deletion, and/or addition of bases to coding sequences of genomic DNAs, which contain at least apart of the nucleotide sequence of SEQ ID NO: 1, to modify the DNAs so as not to encode a polypeptide with trehalase activity, and introducing the modified DNAs into autonomously-replicable vectors. To ease the selection of ES cells in the above step (iii), the targeting vector should preferably be introduced with a sequence as positive and/or negative selective-markers. Concrete examples of the positive selective markers include a neomycin resistant gene and xcex2-galactosidase gene, and those of the negative selective markers include a herpes-simplex-virus-thymidine-kinase gene and a diphtheritic-toxin-A-fragment gene. By using methods such as electroporation, the resulting targeting vector is usually digested with an appropriate restriction enzyme for linearization, and then introduced into animal""s ES cells with a desired trehalase gene, usually, into murine ES cells. When homologous recombination occurs in cells which have been introduced with the targeting vector, the trehalase gene inherent to the cells is destroyed by replacing with a nucleotide sequence which is from the targeting vector and free of encoding a polypeptide with trehalose activity. The desired ES cells can be selected by screening the products from the cells introduced with the vector by using conventional methods such as PCR method for confirming the intracellular homologous recombination (the above step (iii)). With the ES cells thus obtained, the knockout animals of the present invention can be obtained by treating the cells in accordance with the above steps (iv) to (vi). The obtained knockout animals are specifically useful as models for examining the in viva physiological role of trehalase and as animals for screening therapeutic, prophylactic, and diagnostic agents for animal diseases correlated to defection or incompletion of trehalase. The preparation method for knockout animals is called gene targeting and reported in detail in xe2x80x9cNew Genetic Engineering Handbookxe2x80x9d, pp. 277-283 (1996), edited by Masami MURAMATSU, Hiroto OKAYAMA, and Masashi YAMAMOTO, published by Yodo Co., Ltd., Tokyo, Japan.